This invention concerns a nitro-aromatic compound which can be reduced by microorganisms to produce an amino-aromatic compound.
The invention also relates to the use of a compound in a detection and/or diagnostic test for microorganisms.
The invention further relates to a method for demonstrating the presence of nitroaryl reductase activity in a culture medium containing microorganisms.
Finally, the invention covers a method for the detection of single microorganisms or groups of microorganisms in samples which may contain them.
The article xe2x80x9cSyntheses of coumarin derivatives. V Syntheses of coumarin-3-carboxylic acid derivativesxe2x80x9d CHEMICAL ABSTRACTS, vol. 50, 1956, page 10715, XP-002109446, describes the synthesis of nitrocoumarin derivatives including the 7-nitrocoumarins. The only application specified is based on their hypnotic and sedative activities. Moreover, their toxicity is dealt with. The article xe2x80x9cSyntheses of coumarin derivatives. XIV Preparation of 5-hydroxy-7-nitro-3-coumarincarboxylic acidxe2x80x9d CHEMICAL ABSTRACTS, vol. 59, 1963, page 2757, XP-002109447, only describes the synthesis of 5-hydroxy-7-nitrocoumarin carboxylic acid.
The possibility of using these molecules to detect the presence or absence of microorganisms has never been addressed. Therefore, no bacteriological application has ever been contemplated. Moreover, the toxicity results did not encourage those skilled in the art to think about using this type of compound in culture media being used to support the growth of microorganisms.
Certain products synthesized above may have therapeutic applications, in particular those endowed with antibacterial activity. This is documented in the two following articles: the article xe2x80x9cStudies on Synthesis of Coumarin Derivatives. XX. Synthesis and Antibacterial Activity of Derivatives of N-Substituted 3-Coumarincarboxamidexe2x80x9d CHEMICAL AND PHARMACEUTICAL BULLETIN, vol. 16, no. 11. November 1968 (1968-11) pages 2093-2100, XP-002109448, describes the use of nitrocoumarins as antibacterial agents as does also the article xe2x80x9cSynthesis of Coumarin Derivatives. XX. On the Preparation of Ethyl Pyranobenzoxazole carboxylatesxe2x80x9d CHEMICAL AND PHARMACEUTICAL BULLETIN, vol. 14, 1966, pages 1162-1167, XP-002109449.
However, in our invention, the essential property is that nitrocoumarinsxe2x80x94and particularly the 7-nitrocoumarinsxe2x80x94fluoresce when they are in the reduced state. This fluorescence depends on whether certain microorganisms are present or not. It should be noted that the toxicity and antibacterial activity of these compounds could only discourage those skilled in the art from contemplating their use in the detection of microorganisms.
The object of this patent application is completely different. It is not in any way associated with detecting a potentially therapeutic activity such as the inhibition of bacterial growth but, in contrast, is a way of detecting microorganisms through an enzyme activity, namely nitroaryl reductase activity which reduces the non-fluorescent 7-nitrocoumarin (or one of its derivatives) into the fluorescent 7-aminocoumarin (or the corresponding derivative). It therefore concerns a fluorescent test for the universal detection of microorganisms in samples which may contain them and in which the inhibition of bacterial growth is not an issue.
Certain bacteria have been known for many years to be able to reduce aromatic nitro-compounds. From E. coli extracts, Asnis (1957) isolated a flavoprotein which could reduce p-nitrobenzoic acid. Since this seminal finding, nitroaryl reductase activities have been described in various types of microorganism, including obligate aerobes such as Pseudomonas spp. (Won et al. 1974) and Nocardia spp. (Villanueva 1964), obligate anaerobes such as Clostridium spp. (Ancermaier and Simon 1983) and Veillonella spp. (McCormick et al. 1976), fungi (Masuda and Ozaki 1993) and eukaryotic parasites (Douch 1975). A whole range of substrates is known as being reducible by bacterial nitroaryl reductases, especially aromatic nitro-compounds such as p-nitrobenzoic acid, p-nitrophenol, p-nitroaniline and 2,4,6-trinitrotoluene (McCormick et al. 1976).
Although a wide range of different substrates is available, none are suitable for the direct detection of nitroaryl reductase through the production of a fluorescent product.
Therefore, this enzyme activity has to be assayed by indirect methods, e.g. by measuring the disappearance of the substrate or some cofactor. Kitamura et al. (1983), investigating the reduction of methyl p-nitrobenzoate and a series of other aromatic nitro-compounds by E. coli extracts, showed that three distinct and well-defined enzyme activities could be chromatographically isolated on a DEAE-cellulose column, and that each of these distinct three fractions required different cofactors for its activity: the first required NADH; the second required NADPH; and the third required both. Enzyme reactivity was assayed by following the change in optical density (OD) at 340 nanometers (nm) due to the consumption of the NADH and/or the NADPH. Consumption of the NADH and/or the NADPH was associated with the formation of two reaction products, namely methyl p-aminobenzoate and methyl p-hydroxylaminobenzoate. Bryant et al. (1981) also studied E. coli nitroaryl reductases, using nitrofurazone as the substrate. In order to assay enzyme activity, they were able to follow changes in OD at 375 nm (the absorption maximum [xcex max] of nitrofurazone). Using this method, they detected three distinct activities capable of reducing nitrofurazone. The ability of bacteria to reduce nitrofuranes is of great interest in the field of antibacterial chemotherapy (Peterson et al. 1979, Wentzell and McCalla 1980) and it has been shown that the major E. coli nitroaryl reductase (which is NADPH-dependent) is absent in nitrofurazone-resistant mutants (Bryant et al. 1981).
The object of this invention is a fluorescent substrate based on nitrocoumarin which is suitable for the direct detection of nitroaryl reductase activity. When reduced, this type of aromatic nitro-compound gives a product which is fluorescent and therefore easy to detect. The reaction (I) is shown below: 
Unexpectedly, it was found that the vast majority of microorganisms contain nitroreductase activity and are therefore capable of reducing 7-nitrocoumarin derivatives to give a fluorescent productxe2x80x94this is not true of any of the substrates which have been investigated hitherto. Therefore, these derivatives represent a class of universal indicators which make it possible to detect the presence or absence of microorganisms in any given sample.
To this end, this invention concerns a compound for detecting the presence or absence of at least one microorganism. The invention is characterized in that the compound is a nitrocoumarin or one of its derivatives which gives a fluorescent product on reduction.
Particularly, said compound is 7-nitrocoumarin or one of its derivatives.
The compound is characterized by the following structural formula: 
in which R3 is either H or COZ, where Z is conducive to the generation of a ketone, an acid or an ester group, or any other aliphatic group, and in which R4 is either H or a trifluoromethyl (CF3) group or any aliphatic group.
According to a modification, R3 consist of COOCH3, COOC2H5, COOH, COC3H7, CONC4H4O or COCH3 group, and R4 is either H or a CH3 group.
According to another modification, the compound is made up of 7-nitrocoumarin-3-carboxylic acid.
The concentration of 7-nitrocoumarin-3-carboxylic acid ranges from 0.05 to 0.3 mmol/l.
According to a potentially interesting embodiment, the compound described above can be used in a formulation or in combination with one or more other nitrocoumarin compounds or derivatives.
The invention also concerns the use of a compound as defined above in a detection and/or diagnostic test for the presence or absence of microorganisms.
The invention further concerns a first method for detecting single microorganisms or groups of microorganisms in samples which may contain them. This method consists in:
adding to a culture medium containing the sample, at least one nitrocoumarin-based compound, preferably 7-nitrocoumarin or one of its derivatives, and
monitoring for the production of a fluorescent productxe2x80x94the presence or absence of a fluorescent signal corresponding to the presence or absence of the suspected microorganism or group of microorganisms.
The invention further concerns a second method for identifying at least one microorganism in a sample which may contain such microorganisms, comprising the following steps:
adding, in a series of wells, a culture medium containing a single carbon source, such as lactose, glucose, sucrose, etc., an aliquot of the test sample and at least one nitrocoumarin-based compound, preferably 7-nitrocoumarin or one of its derivatives, and
searching each well for the production of a fluorescent productxe2x80x94the presence or absence of this fluorescence over all of the wells enabling the microorganism to be identified. This second method is also called an assimilation test.
Finally, the invention concerns various applications associated with the detection of microbial growth using at least one compound, as defined above, for:
performing a sterility test,
counting the microorganisms present in the sample,
testing the susceptibility of a microorganism to an antimicrobial agent, and
detecting the presence of at least one microorganism.